(a) Field of the Invention
Mechanisms regulating cell proliferation stop and differentiation initiation during the development stage of mammalian embryo, and the proteins involved therein, are provided. More specifically, differentiation regulators, methods of regulating differentiation, transgenic organisms with loss of expression of the differentiation regulator, and methods of preparing the transgenic organisms, are provided.
(b) Description of the Related Art
Homeostasis of regenerative epithelial tissues such as skin and intestine is maintained through a tightly balanced process of proliferation and terminal differentiation. During normal epithelial development, proliferating progenitor cells, often referred to as transiently amplifying (TA) cells, actively divide a limited number of times before they undergo cell-cycle exit and terminally differentiate into postmitotic cells. Cancer can develop as a result of inappropriate proliferation of progenitor cells accompanied by a partial or complete loss of differentiation. Therefore, understanding the signaling networks that control cell-cycle exit and terminal differentiation in epithelial tissues will provide insights into the mechanisms underlying tumorigenesis.
A new signaling network, known as the “Hippo pathway” in Drosophila, seems to be a key developmental program in controlling proliferation and apoptosis for proper organ development in Drosophila. The Ste-20 family kinase Hippo, WW adaptor protein Salvador and NDR kinase Warts are key components of the Hippo pathway that restricts cell proliferation and promotes apoptosis in differentiating epithelial cells by regulating expression of cyclin E and Diap1. The Hippo kinase phosphorylates and activates the Warts kinase, and this process is facilitated by the scaffolding protein Salvador or Mats. Warts, together with Mats, then phosphorylates and inhibits the transcription coactivator Yorkie. Expanded, Merlin and Fat, all of which localize to the plasma membrane, function upstream of the Hippo pathway. In flies, mutations of these factors lead to increased cell proliferation and decreased cell death.
The phenotypes of flies with mutations in the Hippo pathway can be rescued with their respective human counterparts, indicating that the Hippo pathway may play an analogous role as a global regulator of epithelial tissue development in mammals. Several reports on each mammalian component of the Hippo pathway have shown that the pathway is involved in cell death and cell-cycle regulation. MST1/2 kinases (Hippo homologs) were originally reported to be involved in apoptosis with caspase-3-mediated proteolytic activation. LATS1/2 (Warts homologs) have been implicated in the regulation of cell-cycle progression, apoptosis, mitotic exit and cytokinesis. YAP (a Yorkie homolog) has been shown to be involved in apoptosis by interacting with p73. Although mutation of WW45 (a Salvador homolog) has been reported in several cancer cell lines, little is known about the functional significance of WW45 in mammals. So far, only limited biochemical interactions have been reported, including the phosphorylation of LATS1/2 by MST1/2, the association of WW45 with MST1/2 and LATS1/2, binding of LATS1 to MOB1 (a MATS homolog) and formation of a complex comprising RASSF1A, MST2, WW45 and LATS1.
The Hippo pathway has also been implicated in mammalian tumorigenesis. Mice lacking LATS1 develop some types of tumor, and hWW45 and Mats are mutated in several cancer cell lines. NF2, the human ortholog of Merlin, is a tumor-suppressor gene, mutations of which lead to neurofibromatosis. YAP is overexpressed in mammalian cancers and transgenic mice overexpressing YAP have an increased liver size and dysplasia with expanded undifferentiated progenitor cells in the intestine. Of the Hippo pathway proteins, only LATS1-, LATS2-, NF2-, and YAP-null mice have been generated; however, these mice are either early embryonic lethal or fail to recapitulate defects seen in the respective Drosophila mutants. Therefore, compared with Drosophila, much less is known about the physiological function of the Hippo pathway in mammalian epithelial development. Furthermore, the molecular mechanisms by which this pathway is regulated during development are not fully understood in mammals.